1. Field of the Invention
This invention relates to bioelectronic sensors and their use to detect hybridization events occurring in DNA, RNA, PNA and other oligonucleotide systems. In a preferred embodiment the detection of such hybridization events is used to detect and verify an oligonucleotide authentication tag. In another preferred embodiment the bioelectronic sensor incorporates an aptamer which undergoes a detectable change in the presence of a target for which the aptamer is specific.
2. Background Information
The detection of DNA, RNA, nucleic acids comprising base analogs, thiols, etc., and, to a lesser extent, PNA (hereinafter generally referred to as “oligonucleotides” and/or “polynucleotides”) hybridization events is of significant scientific and technological importance, manifested in, for example, the rapidly growing interest in the chip-based characterization of gene expression patterns and the detection of pathogens in both clinical and civil defense settings [Heller, M. J., Annu. Rev. Biomed. Eng. 4, 129-153 (2002)]. Consequently, a variety of optical [Taton, T. A., Mirkin, C. A. & Letsinger, R. L. Science 289, 1757-1760 (2000); Gaylord, B. S., Heeger, A. J. & Bazan, G. C., Proc. Nat. Acad. Sci. USA 99, 10954 (2002); Cao, Y. W. C., Jin, R. C. & Mirkin, C. A., Science 297, 1536-1540 (2002)] and acoustic [Cooper, M. A. et al. Nature Biotech. 19, 833-837 (2001)] detection methods have been proposed.
In these assays one or more target oligonucleotides is brought into proximity to one or more oligonucleotide ligands and hybridization (if any) is detected by noting a change in a detectable “genosensor” moiety such as the presence of a suitable fluorolabel, radiolabel or enzyme label, present on the ligands.
Among these historic genosensors, fluorescence detection methods have historically dominated the state of the art [Heller, M. J., Annu. Rev. Biomed. Eng. 4, 129-153 (2002); Bowtell, D. D. L., Nature Genet. 21, 25-32 (1999); Winzeler, E. A., Schena, M. & Davis, R. W., Methods Enzymol. 306, 3 (1999)].
The application of electronic methods to the sensing of biologically related species has recently been attracting increased attention [Kuhr, W. G., Nature Biotech. 18, 1042-1043 (2000); Willner, I., Science 298, 2407 (2002); Fritz, J., Cooper, E. B., Gaudet, S., Sorger, P. K. & Manalis, S. R. Electronic detection of DNA by its intrinsic molecular charge. Proc. Natl. Acad. Sci., U.S.A. 99, 14142-14146 (2002)].
Advantages of bioelectronic detection include the following:                1. Electrochemical techniques offer the promise of sensitive, rapid and inexpensive screening [Bard, A. J. & Faulkner, L. R. Electrochemical Methods (John W. Willey & Sons, New York, 2001)].        2. Unlike fluorophores that quench or photo-bleach, typical electroactive labels are stable and relatively insensitive to their environment.        3. “Multi-color” labeling is possible by molecular design and synthesis that produce a “spectrum” of derivatives, each having a unique detectable electronic signal [Brazill, S. A., Kim, P. H. & Kuhr, W. G., Anal. Chem. 73, 4882-4890 (2001)].        4. The possibility of mass-production of bioelectronic detectors via the well-developed technical infrastructure of the microelectronics industry, renders electronic detection particularly compatible with microarray-based technologies.        
Oligonucleotides are typically electrochemically silent at moderate applied voltages [Palecek, E. & Jelen, F., Crit. Rev. Anal. Chem. 32, 261-270 (2002)]. The first sequence-selective electronic method for DNA detection was based on the electrochemical interrogation of redox-active intercolators that bind preferentially to double-stranded DNA (dsDNA) [Millan, K. M. & Mikkelsen, S. R., Anal. Chem. 65, 2317-2323 (1993)]. More recently, the sensitivity of this detection approach was improved via electrocatalytic amplification [Kelley, S. O., Boon, E. M., Barton, J. K. & Jackson, N. M. H., Nucleic Acids Res. 27, 4830-4837 (1999)].
In an attempt to reduce high background deriving from the inappropriate binding of hybridization indicators to single-stranded DNA (ssDNA), a “sandwich” type detector has been developed. This approach utilizes an electrode-attached ssDNA sequence that binds the target to the electrode and a second, redox-labeled ligand sequence complimentary to an adjacent sequence on the target [Ihara, T., Maruo, Y., Takenaka, S. & Takagi, M., Nucleic Acids Res. 24, 4273-4280 (1996); Yu, C. J. et al., J. Am. Chem. Soc. 123, 11155-11161 (2001); Umek, R. M. et al., J. Mol. Diag. 3, 74-84 (2001)].
Mirkin and co-workers have developed an electronic DNA detection approach that has demonstrated high sensitivity and selectivity [Park, S. J., Taton, T. A. & Mirkin, C. A., Science 295, 1503-1506 (2002)]. In this resistance-based method, a probe-captured target undergoes a second hybridization event with Au nanoparticle-labeled DNA strands. Subsequent catalytic deposition of silver onto the Au nanoparticles leads to electrical contact and a detectable decrease in the resistance between electrode pairs as an indicator of hybridization.
Despite this interest in electronic oligonucleotide detection, there has been little progress toward the important goal of creating a sensor that is simultaneously sensitive, selective and reagentless (e.g., a sensor obviating further treatment with either hybridization indicators or signaling molecules to yield a detectable indication of hybridization). The “reagentless” feature has been reported in the context of a conjugated polymer-based electrochemical DNA sensor [Korri-Youssoufi, H., Garnier, F., Srivastava, P., Godillot, P. & Yassar, A., J. Am. Chem. Soc. 119, 7388-7389 (1997)]. However, this sensor has only moderate sensitivity due to broad, weakly-defined redox peaks.
More generally, while sensitivity of electronic oligonucleotide sensors of the prior art is impressive (ranging from 0.5 to 32 pM), no electronic sensors have been reported to meet the goal of fM sensitivity. The sensitive sensors require the addition of one or more exogenous reagents.
Recent, high profile examples ranging from geopolitical (e.g., forged documents purporting the solicitation of yellow-cake sales to Iraq) to the medical (e.g. the recent recall of approximately 100,000 bottles of potentially counterfeit LIPITOR® (Atorvastatin Calcium) tablets) are indicative of the growing and increasingly complex risks associated with the counterfeiting of a wide range of documents and materials. Thus motivated, significant research has focused on the development of convenient-yet-unforgeable means of “authentifying” the provenance of documents, drugs and other materials related to medical, industrial, homeland or military security.
The use of DNA as an identifying label was first proposed by Philippe Labacq in U.S. Pat. No. 5,139,812 (issued Aug. 18, 1992). The approach works by concealing coded messages in DNA. Security is provided by the inherent sequence complexity of DNA (Clelland, C. T., Risca, V. and Bancroft, C. Nature 399, 533-534 (1999)).
Existing DNA-based authentication methods, however, have been limited to art, sports memorabilia and other high-value, low-volume applications. More widespread use of the approach has been limited by the cumbersome, time and reagent-intensive methods currently employed for the detection of low concentrations of a target DNA sequence in the presence of orders of magnitude larger background of masking DNA (Clelland, C. T., Risca, V. and Bancroft, C. Nature 399, 533-534 (1999); Cox, J. P. L. Analyst 126, 545-547 (2001)). Unfortunately, the technologies underlying counterfeiting generally keep pace with the technologies aimed at impeding such efforts. Thus, to date, no general, unbreakable means of “authenticating” documents, drugs and other high-volume materials has been reported.
It is the object of this invention to provide an electrochemical method for detecting specific sequences on target oligonucleotides, said method being simultaneously sensitive, selective, reagentless, and reusable. It is a further object to provide an electrochemical method for detecting an oligonucleotide-based (such as, for example, a DNA, RNA or peptide nucleic acid (PNA)-based) authentication tag.
It is a further object of this invention to provide aptamer-based bioelectronic sensors and to enable their use to detect aptamer-specific targets.